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United States Patent | 5,585,102 |
Barbour ,   et al. | December 17, 1996 |
This invention relates to flagella-less strains of Borrelia to novel methods for use of the microorganisms as vaccines and in diagnostic assays. Although a preferred embodiment of the invention is directed to Borrelia burgdorferi, the present invention encompasses flagella-less strains of other microorganisms belonging to the genus Borrelia. Accordingly, with the aid of the disclosure, flagella-less mutants of other Borrelia species, e.g., B. coriacei, which causes epidemic bovine abortion, B. anserina, which causes avian spirochetosis, and B. recurrentis and other Borrelia species causative of relapsing fever, such as Borrelia hermsii, Borrelia turicatae, Borrelia duttoni, Borrelia persica, and Borrelia hispanica, can be prepared and used in accordance with the present invention and are within the scope of the invention. Therefore, a preferred embodiment comprises a composition of matter comprising a substantially pure preparation of a strain of a flagella-less microorganism belonging to the genus Borrelia.
Inventors: | Barbour; Alan G. (San Antonio, TX); Bundoc; Virgilio G. (Newbury Park, CA); Sadziene; Adriadna (San Antonio, TX) |
Assignee: | Board of Regents, The University of Texas System (Austin, TX) |
Appl. No.: | 124290 |
Filed: | September 20, 1993 |
Current U.S. Class: | 424/234.1; 435/7.1; 435/7.2; 435/29; 435/40.5; 435/243; 435/245 |
Intern'l Class: | C12N 001/00; C12N 001/36; A61K 039/02; C12Q 001/02 |
Field of Search: | 424/184.1,185.1,282.1,234.1,93.1,93.2 435/6.7,7.22,243,245,7.1,7.2,29,40.5 |
5187065 | Feb., 1993 | Schitzen | 435/7. |
5217872 | Jun., 1993 | Dorward | 435/7. |
Foreign Patent Documents | |||
9004411 | May., 1990 | WO | . |
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TABLE 1 ______________________________________ Host Cell Association Assay Average cpm (SD) Average % of inoculum Isolate cell associated Cell Associated ______________________________________ W 6880 (342) 5.7 (0.3) M 5173 (276) 4.3 (0.2) R 6256 (336) 5.2 (0.3) Low passage 8208 (425) 6.8 (0.4) HB19 ______________________________________
TABLE 2 ______________________________________ Penetration of Cell Monolayers Average cpm (SD) Average % of inoculum Isolate in lower chamber in lower chamber (SD) ______________________________________ W 33,504 (1144) 6.8 (0.2) M 887 (283) 0.2 (0.1) R 12,810 (569) 2.6 (0.1) Low passage 41,387 (1709) 8.4 (0.4) HB19 ______________________________________
TABLE 3 ______________________________________ ELISA Test With Serum Diluted 1:1,000 Absorbance Rat # Immunogen ELISA antigen Value ______________________________________ 1 W cells W cells 0.713 M cells 0.727 2 W cells W cells 0.592 M cells 0.629 3 M cells W cells 0.555 M cells 0.449 4 M cells W cells 0.589 M cells 1.082 5 Adjuvant alone W cells 0.010 M cells 0.014 6 Adjuvant alone W cells 0.015 M cells 0.013 ______________________________________
TABLE 4 ______________________________________ IMMUNOGEN W cells M cells Adjuvant alone ______________________________________ 1:3,000 1:3,000 .ltoreq.1:10 ______________________________________
__________________________________________________________________________ SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 3 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1507 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Borrelia burgdorferi (B) STRAIN: HB19 (vii) IMMEDIATE SOURCE: (A) LIBRARY: pACA1 (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 207..1217 (D) OTHER INFORMATION: /product="Flagellin" (ix) FEATURE: (A) NAME/KEY: RBS (B) LOCATION: 194..198 (ix) FEATURE: (A) NAME/KEY: 10_signal (B) LOCATION: 146..151 (ix) FEATURE: (A) NAME/KEY: -35_signal (B) LOCATION: 122..125 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: CCTTGGATTTTACCGTTAAGCGCATGAAAGATCAAGAAAATACATTAAAGGCTTTTGATT60 TTAATCAAAGAAATAAATAATAATAATTATTTTTAATGCCATTGCTATCTGCGTTTCTTT120 TTTTTAAATTTTTGTGCTATTCTTTTTAACAGGCAAAAGGATTTGCCAAAACCAAATAAT180 TAAATTTTATCATGGAGGAATGATATATGATTATCAATCATAATACATCAGCT233 MetIleIleAsnHisAsnThrSerAla 15 ATTAATGCTTCAAGAAATAATGGCATTAACGCTGCTAATCTTAGTAAA281 IleAsnAlaSerArgAsnAsnGlyIleAsnAlaAlaAsnLeuSerLys 10152025 ACTCAAGAAAAGCTTTCTAGTGGGTACAGAATTAATCGAGCTTCTGAT329 ThrGlnGluLysLeuSerSerGlyTyrArgIleAsnArgAlaSerAsp 303540 GATGCTGCTGGCATGGGAGTTTCTGGTAAGATTAATGCTCAAATAAGA377 AspAlaAlaGlyMetGlyValSerGlyLysIleAsnAlaGlnIleArg 455055 GGTTTGTCACAAGCTTCTAGAAATACTTCAAAGGCTATTAATTTTATT425 GlyLeuSerGlnAlaSerArgAsnThrSerLysAlaIleAsnPheIle 606570 CAGACAACAGAAGGGAATTTAAATGAAGTAGAAAAAGTCTTAGTAAGA473 GlnThrThrGluGlyAsnLeuAsnGluValGluLysValLeuValArg 758085 ATGAAGGAATTGGCAGTTCAATCAGGTAACGGCACATATTCAGATGCA521 MetLysGluLeuAlaValGlnSerGlyAsnGlyThrTyrSerAspAla 9095100105 GACAGAGGTTCTATACAAATTGAAATAGAGCAACTTACAGACGAAATT569 AspArgGlySerIleGlnIleGluIleGluGlnLeuThrAspGluIle 110115120 AATAGAATTGCTGATCAAGCTCAATATAACCAAATGCACATGTTATCA617 AsnArgIleAlaAspGlnAlaGlnTyrAsnGlnMetHisMetLeuSer 125130135 AACAAATCTGCTTCTCAAAATGTAAGAACAGCTGAAGAGCTTGGAATG665 AsnLysSerAlaSerGlnAsnValArgThrAlaGluGluLeuGlyMet 140145150 CAGCCTGCAAAAATTAACACACCAGCATCACTTTCAGGGTCTCAAGCG713 GlnProAlaLysIleAsnThrProAlaSerLeuSerGlySerGlnAla 155160165 TCTTGGACTTTAAGAGTTCATGTTGGAGCACCCCAAGATGAAGCTATT761 SerTrpThrLeuArgValHisValGlyAlaProGlnAspGluAlaIle 170175180185 GCTGTAAATATTTATGCAGCTAATGTTGCAAATCTTTTCTCTGGTGAG809 AlaValAsnIleTyrAlaAlaAsnValAlaAsnLeuPheSerGlyGlu 190195200 GGAGCTCAAACTGCTCAGGCTGCACCGGTTCAAGAGGGTGTTCAACAG857 GlyAlaGlnThrAlaGlnAlaAlaProValGlnGluGlyValGlnGln 205210215 GAAGGAGCTCAACAGCCAGCACCTGCTACAGCACCTTCTCAAGGCGGA905 GluGlyAlaGlnGlnProAlaProAlaThrAlaProSerGlnGlyGly 220225230 GTTAATTCTCCTGTTAATGTTACAACTACAGTTGATGCTAATACATCA953 ValAsnSerProValAsnValThrThrThrValAspAlaAsnThrSer 235240245 CTTGCTAAAATTGAAAATGCTATTAGAATGATAAGTGATCAAAGAGCA1001 LeuAlaLysIleGluAsnAlaIleArgMetIleSerAspGlnArgAla 250255260265 AATTTAGGTGCTTTCCAAAATAGACTTGAATCTATAAAGGATAGTACT1049 AsnLeuGlyAlaPheGlnAsnArgLeuGluSerIleLysAspSerThr 270275280 GAGTATGCAATTGAAAATCTAAAAGCATCTTATGCTCAAATAAAAGAT1097 GluTyrAlaIleGluAsnLeuLysAlaSerTyrAlaGlnIleLysAsp 285290295 GCTACAATGACAGATGAGGTTGTAGCAGCAACAACTAATAGTATTTTA1145 AlaThrMetThrAspGluValValAlaAlaThrThrAsnSerIleLeu 300305310 ACACAATCTGCAATGGCAATGATTGCGCAGGCTAATCAAGTTCCCCAA1193 ThrGlnSerAlaMetAlaMetIleAlaGlnAlaAsnGlnValProGln 315320325 TATGTTTTGTCATTGCTTAGATAAAATTCTAGTTTAATTGGTATAAATTGT1244 TyrValLeuSerLeuLeuArg 330335 ATTAACAAAAGATCCTTTAAAGGATCTTTTGTTTTTTTATTTCAGATCGGCAAAAATTTA1304 ATAATTTTAGTATAATTTATAATGATGTGTTATAAAGCCATAATGCAGGCTTATTGAGGA1364 GTTTGCTTTGAGTAGGGTTGCTGTTAAAATTAAAGATTGTTATAAAGTATTTTCGTATTA1424 TGTTAACAAAAAGCAGATAAGTCGAGCTATAAAAGACTATGAAGATGGCAAAGATAGAAT1484 GCAAATCTACAAAGAATCTTCTA1507 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 336 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: MetIleIleAsnHisAsnThrSerAlaIleAsnAlaSerArgAsnAsn 151015 GlyIleAsnAlaAlaAsnLeuSerLysThrGlnGluLysLeuSerSer 202530 GlyTyrArgIleAsnArgAlaSerAspAspAlaAlaGlyMetGlyVal 354045 SerGlyLysIleAsnAlaGlnIleArgGlyLeuSerGlnAlaSerArg 505560 AsnThrSerLysAlaIleAsnPheIleGlnThrThrGluGlyAsnLeu 65707580 AsnGluValGluLysValLeuValArgMetLysGluLeuAlaValGln 859095 SerGlyAsnGlyThrTyrSerAspAlaAspArgGlySerIleGlnIle 100105110 GluIleGluGlnLeuThrAspGluIleAsnArgIleAlaAspGlnAla 115120125 GlnTyrAsnGlnMetHisMetLeuSerAsnLysSerAlaSerGlnAsn 130135140 ValArgThrAlaGluGluLeuGlyMetGlnProAlaLysIleAsnThr 145150155160 ProAlaSerLeuSerGlySerGlnAlaSerTrpThrLeuArgValHis 165170175 ValGlyAlaProGlnAspGluAlaIleAlaValAsnIleTyrAlaAla 180185190 AsnValAlaAsnLeuPheSerGlyGluGlyAlaGlnThrAlaGlnAla 195200205 AlaProValGlnGluGlyValGlnGlnGluGlyAlaGlnGlnProAla 210215220 ProAlaThrAlaProSerGlnGlyGlyValAsnSerProValAsnVal 225230235240 ThrThrThrValAspAlaAsnThrSerLeuAlaLysIleGluAsnAla 245250255 IleArgMetIleSerAspGlnArgAlaAsnLeuGlyAlaPheGlnAsn 260265270 ArgLeuGluSerIleLysAspSerThrGluTyrAlaIleGluAsnLeu 275280285 LysAlaSerTyrAlaGlnIleLysAspAlaThrMetThrAspGluVal 290295300 ValAlaAlaThrThrAsnSerIleLeuThrGlnSerAlaMetAlaMet 305310315320 IleAlaGlnAlaAsnGlnValProGlnTyrValLeuSerLeuLeuArg 325330335 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GCCAGCAGCATCATCAGAAG20 __________________________________________________________________________